Analysis device and analysis method

ABSTRACT

An analysis device optically scans a surface of a substrate to which particles are fixed, detects a pulse wave included in a detection signal obtained from an optical scanning unit when the optical scanning unit scans the substrate, and counts the particles based on pulse interval between two pulse waves each having pulse width less than first reference value determined depending on first pulse width when the optical scanning unit scans a plurality of particles adjacent to each other when the two pulse waves are detected consecutively.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of PCT Application No.PCT/JP2015/057303, filed on Mar. 12, 2015, and claims the priority ofJapanese Patent Application No. 2014-072536, filed on Mar. 31, 2014, theentire contents of both of which are incorporated herein by reference.

BACKGROUND

The present disclosure relates to an analysis device and an analysismethod for analyzing biomaterials such as antibodies and antigens.

Immunoassays are known that quantitatively analyze disease detection andtherapeutic effects by detecting particular antigens or antibodies asbiomarkers associated with diseases. One of the immunoassays is anenzyme-linked immunosorbent assay (ELISA) for detecting antigens orantibodies labeled by enzymes, which is widely used because of havingthe advantage of low costs. The ELISA requires a long period of time,such as from several hours to a day, to complete a series of multiplesteps including pretreatment, antigen-antibody reaction, bond/free (B/F)separation, and enzyme reaction.

Another technology is disclosed in which antibodies fixed to an opticaldisc are allowed to bind to antigens in a specimen, and the antigens arefurther bound to particles having antibodies and then scanned with anoptical head, so as to count the particles captured on the disc in ashort period of time (Japanese Unexamined Patent Application PublicationNo. H05-005741). Still another technology is disclosed in whichbiosamples or particles are adsorbed to a surface of an optical disc onwhich a tracking structure is formed, so as to detect changes in signalby an optical pickup (Japanese Translation of PCT InternationalApplication Publication No. 2002-530786).

SUMMARY

The technology disclosed in Japanese Unexamined Patent ApplicationPublication No. H05-005741 or Japanese Translation of PCT InternationalApplication Publication No. 2002-530786, however, may fail to obtaindetection signals corresponding to particles depending on the type andarrangement of the particles used. Such failure leads to inaccuratecounting results, which may decrease the performance of quantitativeanalysis of analytes.

A first aspect of the present embodiment provides an analysis deviceincluding: an optical scanning unit configured to optically scan asurface of a substrate to which particles are fixed; a pulse detectorconfigured to detect a pulse wave and a pulse width of the pulse waveincluded in a detection signal obtained from the optical scanning unitwhen the optical scanning unit scans the substrate; and a counting unitconfigured to count the particles based on a pulse interval between twopulse waves each having a pulse width less than a first reference valuewhen the pulse detector consecutively detects the two pulse waves.

A second aspect of the present embodiment provides an analysis methodincluding: optically scanning a surface of a substrate to whichparticles are fixed; detecting a pulse wave and a pulse width of thepulse wave included in a detection signal obtained by scanning thesubstrate; and counting the particles based on a pulse interval betweentwo pulse waves each having a pulse width less than a first referencevalue when the two pulse waves are consecutively detected in thedetection signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram for describing a fundamentalconfiguration of an analysis device according to an embodiment.

FIG. 2A to FIG. 2F are enlarged cross-sectional views each schematicallyshowing a substrate of the analysis device according to the embodiment,for describing an example of a method of fixing antibodies, antigens,and beads to the substrate.

FIG. 3 is a view showing simulation results of detection of adjacentbeads while varying the number of beads, for describing characteristicsbetween a spot position and signal intensity when scanning thesubstrate.

FIG. 4 is a view for describing characteristics between a spot positionand signal intensity when scanning the substrate of the analysis deviceaccording to the embodiment, in which adjacent beads are detected whilevarying the number of beads.

FIG. 5 is a view for describing characteristics between a spot positionand signal intensity when scanning the substrate of the analysis deviceaccording to the embodiment, in which adjacent beads are detected whilevarying the number of beads.

FIG. 6 is a view for describing a method of determining a referencevalue stored in a storage unit included in the analysis device accordingto the embodiment.

FIG. 7 is a view for describing a method of determining a referencevalue stored in the storage unit included in the analysis deviceaccording to the embodiment.

FIG. 8 is a flowchart for describing the operation of the analysisdevice according to the embodiment.

FIG. 9 is a view for describing the operation of the analysis deviceaccording to the embodiment.

FIG. 10 is a view for describing the operation of the analysis deviceaccording to the embodiment.

FIG. 11 is a view for comparing characteristics of biomarkerconcentration and counting results of beads in the analysis deviceaccording to the embodiment with those in a conventional device.

DETAILED DESCRIPTION

Hereinafter, an embodiment will be described with reference to thedrawings. The same or similar elements shown in the drawings aredesignated by the same or similar reference numerals below, andoverlapping descriptions thereof are not repeated herein.

Analysis Device

As shown in FIG. 1, an analysis device according to the embodimentincludes a substrate 100, a motor 2 that rotates the substrate 100, anoptical scanning unit 3 that optically scans the substrate 100, and acontroller 5 that controls the motor 2 and the optical scanning unit 3.

The substrate 100 is formed into a circular shape having substantiallythe same dimensions as optical discs such as compact discs (CDs),digital versatile discs (DVDs), and Blu-ray discs (BD). The substrate100 has a track structure on the surface thereof that the opticalscanning unit 3 can scan. The track structure includes, for example,grooves, lands, and pits, and is formed into a spiral extending from theinner side to the outer side. The substrate 100 is formed of ahydrophobic resin material, such as polycarbonate resin and cycloolefinpolymer, used for common optical discs. The substrate 100 may be, asnecessary, provided with a thin film on the surface thereof, orsubjected to surface treatment with a silane coupling agent.

As shown in FIG. 2A, antibodies 61 specifically binding to antigens 62,which are biomaterials serving as analytes, are fixed to the surface ofthe substrate 100. The antigens 62 are labeled with beads (particles) 66to which antibodies 65 specifically binding to the antigens 62 areadsorbed, so that the antigens 62 and the beads 66 are correlativelyfixed to the surface of the substrate 100. The antigens 62 arespecifically bound to the antibodies 61 and 65, so as to be used asbiomarkers serving as indicators of diseases.

As shown in FIG. 2A, the antibodies 61 are preliminarily fixed to thesurface of the substrate 100. The antibodies 61 are bound to the surfaceof the substrate 100 due to hydrophobic binding or covalent binding. Theantibodies 61 may be fixed to the surface of the substrate 100 via asubstance such as avidin. Then, as shown in FIG. 2B, a sample solution63 including the antigens 62 is applied dropwise to the surface of thesubstrate 100. The antigens 62 move through the sample solution 63 byBrownian motion and come into contact with the antibodies 61, so as tobe specifically bound to the antibodies 61 by an antigen-antibodyreaction. As shown in FIG. 2C, the surface of the substrate 100 to whichthe sample solution 63 is applied dropwise is subjected to spin washingwith pure water or the like, so as to remove the sample solution 63including excessive antigens 62 not bound to the antibodies 61 from thesurface of the substrate 100.

As shown in FIG. 2D, a buffer solution 64 including the beads 66 isapplied dropwise to the surface of the substrate 100. The buffersolution 64 may be applied while the sample solution 63 remains on thesurface of the substrate 100. The antibodies 65 adsorbed to the beads 66specifically bind to the antigens 62 by the antigen-antibody reaction.The beads 66 are then bound to the antigens 62, so as to label theantigens 62.

The beads 66 are formed of synthetic resin such as polystyrene includinga magnetic material such as ferrite, and formed into a substantiallyspherical shape. A diameter of the beads 66 is in the range of fromseveral tens of nanometers to several hundreds of nanometers, and aparticular example of the diameter is 200 nm. When the buffer solution64 is applied dropwise, the beads 66 are quickly collected to thesurface of the substrate 100 such that a magnet is placed on theopposite side of the surface of the substrate 100, so as to promote thereaction with the antigens 62. In addition, the time required to labelthe antigens 62 fixed to the substrate 100 can be reduced toapproximately several minutes such that the antigens 62 and the beads 66are simultaneously applied to the substrate 100.

The antibodies 61 and 65 may be any biomaterials having specificity thatspecifically bind to the antigens 62. A combination of the antibodies 61and 65 is selected such that the antibodies 61 and 65 separately bind todifferent sites. For example, when membrane vesicles such as exosomes onwhich several types of antigens 62 are expressed are used as analytes,the types of the antibodies 61 and 65 are chosen differently from eachother, so as to detect a biosample including two types of antigens 62.The antibodies 61 and 65 are, however, not limited thereto, and may bethe same type because exosomes, which are different from typicalantigens, include multiple antigens of the same kind of protein on thesurface thereof.

As shown in FIG. 2E, the substrate 100 to which the buffer solution 64is applied dropwise is washed with, for example, pure water, so as toremove the buffer solution 64 including excessive beads 66 not bound tothe antigens 62 from the substrate 100. As shown in FIG. 2F, thesubstrate 100 is optically scanned by the optical scanning unit 3, so asto detect the beads 66 to analyze the antigens 62 labeled with the beads66.

As shown in FIG. 1, the optical scanning unit 3 includes a laseroscillator 31, a collimator lens 32, a beam splitter 33, an actuator 34,an objective lens 35, a condensing lens 36, and a light detector 37 .The optical scanning unit 3 is an optical pickup that optically scansthe substrate 100.

The laser oscillator 31 emits laser light to the collimator lens 32according to the control by the controller 5. The laser oscillator 31 isa semiconductor laser oscillator that emits laser light having, forexample, a wavelength of 405 nm which is the same as that forreproduction of BD, and output of about 1 mW. The collimator lens 32collimates the laser light emitted from the laser oscillator 31. Thebeam splitter 33 reflects the laser light collimated by the collimatorlens 32 toward the objective lens 35.

The objective lens 35 concentrates the laser light transmitted via thebeam splitter 33 on the surface of the substrate 100, to which theantibodies 61 are fixed, due to the operation of the actuator 34according to the control by the controller 5, so as to image spot S. Theobjective lens 35 has a numerical aperture of, for example, 0.85. Thelaser light concentrated by the objective lens 35 is reflected from thesubstrate 100 and then reaches the beam splitter 33. The incident laserlight passes through the beam splitter 33 and further reaches the lightdetector 37 via the condensing lens 36. The condensing lens 36concentrates the laser light reflected from the substrate 100 into thelight detector 37. The light detector 37 is, for example, a photodiodeto output, to the controller 5, a detection signal corresponding to thevolume of the laser light reflected from the substrate 100.

The controller 5 controls the operation of the motor 2 via a rotationcontroller 21. The motor 2 is controlled by the controller 5 to rotatethe substrate 100 at a constant linear velocity (CLV). The linearvelocity is, for example, 4.92 m/s.

The controller 5 controls the operation of the laser oscillator 31 andthe actuator 34 via an optical system controller 4. The actuator 34 iscontrolled by the controller 5 to move the optical scanning unit 3 in aradial direction of the substrate 100 so as to spirally scan the surfaceof the rotating substrate 100. The controller 5 also detects errors suchas focus errors (FE) or tracking errors (TE) from the detection signaloutput from the light detector 37. The controller 5 controls theactuator 34 and other components to appropriately scan the surface ofthe substrate 100 depending on the errors detected.

The controller 5 includes a pulse detector 51, a storage unit 52, and acounting unit 50. The pulse detector 51 inputs the detection signaloutput from the light detector 37. The pulse detector 51 detects a pulsewave and a pulse width of the pulse wave included in the detectionsignal obtained from the optical scanning unit 3. The pulse detector 51is a signal processing device such as a digital signal processor (DSP).The storage unit 52 is a memory such as a semiconductor memory. Thestorage unit 52 stores reference values corresponding to the pulse waveand the pulse width detected by the pulse detector 51.

The counting unit 50 counts the number of beads 66 fixed to the surfaceof the substrate 100 according to the pulse wave detected by the pulsedetector 51 and the reference values stored in the storage unit 52. Thecounting unit 50 is, for example, a central processing unit (CPU). Thecounting unit 50 includes, as a logical structure, a first counter 501,a second counter 502, and a target counter 503.

The first counter 501 measures pulse width Ta of the pulse wave detectedby the pulse detector 51. The second counter 502 measures, depending onthe pulse width Ta of the pulse wave detected by the pulse detector 51,pulse interval Tb between the pulse wave and a pulse wave subsequentlydetected. The target counter 503 counts the number of beads 66 accordingto the measurement results by the first counter 501 and the secondcounter 502 and the reference values stored in the storage unit 52.

Reference Values

FIG. 3 shows simulation results of three detection signals DS1 to DS3obtained in such a manner as to scan each of one projection pit assumingthat one bead is present on the substrate 100, adjacent two projectionpits assuming that two beads are present on the substrate 100, andadjacent three projection pits assuming that three beads are present onthe substrate 100. The transverse axis represents a position of the spotS corresponding to each front bead 66 in a particular interval, and thevertical axis represents signal intensity obtained such that each signalis normalized by a detection signal detected when there is no bead 66.The pulse width is assumed to gradually increase as the number of beads66 increases, as indicated by the detection signal DS1 with one isolatedbead 66, the detection signal DS2 with two beads 66, and the detectionsignal DS3 with three beads 66 in this order.

As shown in FIG. 4, however, a detection signal D2 obtained such thatadjacent two beads 66 are actually scanned includes two pulse waves withsubstantially the same pulse width which is smaller than a pulse widthof a detection signal D1 obtained such that one isolated bead 66 isscanned. As shown in FIG. 5, a detection signal D3 obtained such thatadjacent three beads are actually scanned includes two pulse waveshaving substantially the same pulse width as those of the detectionsignal D2 and having a larger pulse interval than the detection signalD2. A detection signal with adjacent four beads 66 scanned also includestwo pulse waves having substantially the same width as those of thedetection signal D2 and having a larger pulse interval than thedetection signal D3.

When the diameter of beads 66 is approximately one half of thewavelength of the laser light scanned, and there are a plurality ofbeads 66 adjacent to each other, the number of beads cannot be countedaccurately, which may decrease the performance of quantitative analysisof the analytes. The inventors resolved the effects of light on astructure (particles) with a smaller size than a wavelength of the lighthaving different pits from common optical discs as described above, bysolving Maxwell's equations with regard to times and space variables bya finite-difference time-domain (FDTD) method. The counting unit 50 cancount the number of beads 66 with high accuracy when a plurality ofbeads 66 adjacent to each other are present on the substrate 100, on thebasis of the predetermined reference values stored in the storage unit52.

As shown in FIG. 6, the storage unit 52 preliminarily stores first pulsewidth T1 of the respective detection signals D2 and D3 and firstreference value T2 determined depending on the first pulse width T1 whenthe optical scanning unit 3 scans a plurality of beads 66 adjacent toeach other. The first reference value T2 is, for example, the sum of thefirst pulse width T1 and a predetermined value. The predetermined valueadded to the first pulse width T1 maybe a jitter value included in thedetection signal. The predetermined value added to the first pulse widthT1 may also be approximately 100% to 130% of the jitter value. The firstreference value T2 may be a predetermined percentage of the first pulsewidth T1. For example, the first reference value T2 is approximately100% to 130% of the first pulse width T1.

As shown in FIG. 7, the storage unit 52 preliminarily stores secondpulse width T3 of the detection signal Dl and second reference value T4determined depending on the second pulse width T3 when the opticalscanning unit 3 scans a bead 66 isolated from other beads 66. The secondreference value T4 is, for example, the sum of the second pulse width T3and a predetermined value. The predetermined value added to the secondpulse width T3 may be a jitter value included in the detection signal.The predetermined value added to the second pulse width T3 may also beapproximately 100% to 130% of the jitter value. The second referencevalue T4 may be a predetermined percentage of the second pulse width T3.For example, the second reference value T4 is approximately 100% to 130%of the second pulse width T3.

Analysis Method

An analysis method by the analysis device according to the embodiment isdescribed below with reference to the flowchart shown in FIG. 8, inwhich the optical scanning unit 3 optically scans the substrate 100, andthe counting unit 50 counts the number of beads 66 fixed to thesubstrate 100, so as to analyze the analytes labeled by the beads 66.

First, the operator allows the rotation controller 21 and the opticalsystem controller 4 to respectively start operations of the motor 2 andthe optical scanning unit 3 according to the control by the controller5. The substrate 100 to which antigens 62 and beads 66 are fixed on thesurface thereof by the antigen-antibody reaction, is rotated at aconstant linear velocity by the motor 2, so as to be optically scannedby the optical scanning unit 3. The optical scanning unit 3 detects,with the light detector 37, the laser light emitted from the laseroscillator 31 and reflected from the surface of the substrate 100. Thelight detector 37 outputs a detection signal corresponding to the volumeof the detected laser light to the pulse detector 51.

In step S1, the pulse detector 51 obtains the detection signal outputfrom the light detector 37 to detect a falling edge of the obtaineddetection signal. The pulse detector 51 preliminarily holds a thresholdset to intensity corresponding to approximately one half of a peak valueof the detection signal detected when beads 66 are scanned, and detectsa point where the detection signal falls below the threshold as afalling edge of the detection signal.

In step S2, the first counter 501 starts measuring time Ta from thepoint where the falling edge is detected in step S1, as shown in FIG. 9.

In step S3, the pulse detector 51 detects a rising edge of the detectionsignal obtained from the light detector 37. The pulse detector 51preliminarily holds the threshold set to the intensity corresponding toapproximately one half of the peak value of the detection signaldetected when beads 66 are scanned, and detects a point where thedetection signal exceeds the threshold as a rising edge of the detectionsignal.

In step S4, the first counter 501 fixes the time Ta from the point wherethe falling edge is detected in step S1 to the point where the risingedge is detected in step S3, and resets it. The target counter 503obtains and holds the time Ta fixed by the first counter 501 as a pulsewidth (half width) Ta of the pulse wave detected in steps S1 to S3.

In step S5, the target counter 503 reads out the first reference valueT2 from the storage unit 52, and determines whether the pulse width Taheld in step S4 is less than the first reference value T2. The targetcounter 503 sets the process proceeding to step S6 when the pulse widthTa is less than the first reference value T2, or sets the processproceeding to step S9 when the pulse width Ta is greater than or equalto the first reference value T2.

When the pulse width Ta is less than the first reference value T2 instep S5, the target counter 503 determines whether an adjacent flag is“High” (=1) in step S6. The adjacent flag is a flag set in the targetcounter 503 in association with the second counter 502. The targetcounter 503 sets the process proceeding to step S7 when the adjacentflag is “High” in step S6, or sets the process proceeding to step S12when the adjacent flag is “Low” (=0).

In the example shown in FIG. 9, it is assumed that the detection signalD3 is input into the pulse detector 51, and the pulse detector 51detects the rising edge of the first pulse wave in step S3. In such acase, since the adjacent flag is “Low” in step S6, the counting unit 50sets the process proceeding to step S12.

In step S12, the second counter 502 starts measuring time Tb from thepoint where the rising edge is detected in step S3. The target counter503 sets, in association with the second counter 502, the adjacent flagto “High” from the point where the rising edge is detected in step S3,and the process proceeds to step S8.

In step S8, the controller 5 determines whether the scanning of thesubstrate 100 in a predetermined tracking range by the optical scanningunit 3 is finished. The controller 5 ends the process when the scanningis finished, or sets the process returning to step S1 when the scanningis not yet finished.

In the example shown in FIG. 9, it is assumed that the detection signalD3 is input into the pulse detector 51, and the pulse detector 51detects the rising edge of the second pulse wave in step S3. In such acase, since the adjacent flag is “High” in step S6, the counting unit 50sets the process proceeding to step S7.

In step S7, the second counter 502 determines the time Tb from the pointwhere the first rising edge is detected in step S3 to the point wherethe second rising edge is detected in the next step S3, and resets it.The target counter 503 obtains and holds the time Tb determined by thesecond counter 502 as a pulse interval Tb of the two pulse wavesdetected in the two sets of steps S1 to S3, and sets the adjacent flagto “Low”.

In step S7, the target counter 503 determines that the optical scanningunit 3 has scanned a plurality of beads 6 adjacent to each other, so asto read out the first pulse width T1 from the storage unit 52 to countthe number of beads 66 according to “1+(Tb/T1)”. The value obtained from(Tb/T1) is, for example, rounded off to the nearest integer. In theexample shown in FIG. 9, when the detection signal D3 is input into thepulse detector 51, the number of beads 66 results in 1+2=3. As describedabove, when the two pulse waves each having the pulse width Ta less thanthe first reference value T2 are detected consecutively, the targetcounter 503 counts the number of beads 66 according to “1+(Tb/T1)”. Thetarget counter 503 sets the process proceeding to step S8 after step S7.

When the pulse width Ta is greater than or equal to the first referencevalue T2 in step S5, the target counter 503 reads out the secondreference value T4 from the storage unit 52, and determines in step S9whether the pulse width Ta held in step S4 is less than the secondreference value T4. The target counter 503 sets the process proceedingto step S10 when the pulse width Ta is less than the second referencevalue T4, or sets the process proceeding to step S11 when the pulsewidth Ta is greater than or equal to the second reference value T4.

As shown in the example of FIG. 10, it is assumed that the detectionsignal D1 is input into the pulse detector 51, and the pulse detector 51detects the rising edge of the pulse wave in step S3. In such a case,since the pulse width Ta is greater than or equal to the first referencevalue T2 in step S5, and the pulse width Ta is less than the secondreference value T4 in step S9, the counting unit 50 sets the processproceeding to step S10.

In step S10, the target counter 503 determines that the optical scanningunit 3 has scanned one bead 66 isolated from other beads 66 andtherefore the count of beads 66 results in one. Thus, the target counter503 determines that the number of beads 66 counted is one when the pulsewave having the pulse width Ta greater than or equal to the firstreference value T2 and less than the second reference value T4 isdetected. The target counter 503 then sets the adjacent flag to “Low”,and the process proceeds to step S8.

When the pulse width Ta is greater than or equal to the second referencevalue T4 in step S9, the target counter 503 determines in step S11 thatthe pulse wave having the pulse width greater than or equal to thesecond reference value T4 is noise derived from foreign substances oraggregations, so as not to consider the pulse wave when implementingcounting processing. The target counter 503 then sets the adjacent flagto “Low”, and the process proceeds to step S8.

It is also assumed that the pulse wave having the pulse width Ta lessthan the first reference value T2 is detected in the first set of stepsS1 to S3, and the pulse wave having the pulse width Ta greater than orequal to the first reference value T2 and less than the second referencevalue T4 is detected in the next set of steps S1 to S3. In such a case,the target counter 503 determines that the pulse wave detected first isnoise derived from foreign substances or aggregations, so as not toconsider the pulse wave when implementing counting processing.

As described above, when the pulse wave having the pulse width Ta lessthan the first reference value T2 is detected in the detection signal,the target counter 503 adds the number based on the pulse width Ta andthe first pulse width T1 to count up the number of beads 66. When thepulse wave having the pulse width Ta greater than or equal to the firstreference value T2 and less than the second reference value T4 isdetected in the detection signal, the target counter 503 adds 1 to countup the number of beads 66.

Comparative Example

A comparative example in which the counted results of beads 66 obtainedby the analysis device according to the embodiment are compared with thecounted results obtained by a conventional method, is described belowwith reference to FIG. 11. The transverse axis represents biomarkerconcentration of analytes, and the vertical axis represents the countedresults of beads 66. The counted results obtained by the analysis deviceaccording to the embodiment are indicated by the curved line P1, and thecounted results obtained by the conventional method are indicated by thecurved line P2.

The analysis revealed that the count is entirely smaller in the curvedline P2 than the curved line P1 regardless of the biomarkerconcentration, in which the maximum difference therebetween is severaltens of percent. As indicated by the broken lines along the respectivecurved lines P1 and P2, when the biomarker content is zero, the countwould ideally result in zero. In the detection method by use of theantigen-antibody reaction, however, nonspecific adsorption appears onthe substrate 100 other than the binding by the antigen-antibodyreaction. Even when the biomarker concentration is zero, the beads 66fixed to the surface of the substrate 100 due to the nonspecificadsorption are thus inevitably counted.

In the respective curved lines P1 and P2, the points of contact (pointsof intersection) between the lower limits of error and the backgroundnoise level Q of the respective curved lines P1 and P2 are respectivelydenoted by the limits of detection R1 and R2. The limit of detection R1in the analysis device according to the embodiment is improved comparedwith the limit of detection R2 in the conventional method, and it isapparent that the sensitivity of the biomarker detection is improved.Accordingly, the analysis device according to the embodiment can improvethe sensitivity for detecting diseases.

The analysis device according to the embodiment varies the number to beadded depending on the pulse width of the detection signal to count upthe beads 66 when a plurality of beads 66 adjacent to each other arefixed onto the substrate 100. Therefore, the analysis device accordingto the embodiment can count the beads 66 with high accuracy to improvethe quantitative analysis of analytes even when irregular pulse wavesare detected in the detection signal because of arrangement of the beads66.

Further, since the first reference value T2 and the second referencevalue T4 are determined in view of the jitter value of the detectionsignal, the analysis device according to the embodiment can count thebeads 66 with higher accuracy, so as to reduce the influence of jitterwhen classifying the pulse width Ta.

Other Embodiments

While the present invention has been described above by reference to theembodiment, the present invention is not intended to be limited to thedescriptions and drawings which form part of the disclosure. Variousalternative embodiments, examples, and practical applications will beapparent to those skilled in the art from this disclosure.

For example, in the embodiment described above, the combination of thebiomaterials as analytes and specific biomaterials specifically bindingto the analytes is not limited to the combination of the antigens 62 andthe antibodies and antibodies 65 fixed to the beads 66. Examples ofspecifically-binding combinations include a combination of a ligand andan acceptor (such as enzymatic proteins, lectins, and hormones), and acombination of nucleic acids having complementary base sequences to eachother.

Alternatively, a well formed of, for example, silicone rubber may beprovided on the surface of the substrate 100, and the reaction betweenthe target antibodies 61, antigens 62 and beads 66 and the removal ofmaterials not reacted by washing may be implemented within the well, soas to exclude the steps of, for example, spin washing and drying tosimplify the process. Further, a plurality of wells may be provided inthe same radius within the allowable area of the substrate 100, so as tomeasure a plurality of specimens simultaneously.

The present invention includes a program for executing, by a computer,the functions of a notifying device according to the embodimentdescribed above. The program may be read out from a storage medium andinput into the computer, or may be transmitted via an electricalcommunication circuit and input into the computer.

The present invention, of course, includes other embodiments notdescribed in this description, such as embodiments including theabove-described configurations mutually applied. Therefore, the scope ofthe present invention is defined only by the appropriate featuresaccording to the claims in view of the explanations made above.

What is claimed is:
 1. An analysis device comprising: an opticalscanning unit configured to optically scan a surface of a substrate towhich particles are fixed; a pulse detector configured to detect a pulsewave and a pulse width of the pulse wave included in a detection signalobtained from the optical scanning unit when the optical scanning unitscans the substrate; and a counting unit configured to count theparticles based on a pulse interval between two pulse waves each havinga pulse width less than a first reference value when the pulse detectorconsecutively detects the two pulse waves.
 2. The analysis deviceaccording to claim 1, wherein, when the two pulse waves each having thepulse width less than the first reference value are consecutivelydetected by the pulse detector, the counting unit counts the particlesbased on a number obtained by dividing the pulse interval between thetwo pulse waves by a first pulse width detected when the opticalscanning unit scans the particles adjacent to each other.
 3. Theanalysis device according to claim 2, wherein the first reference valueis a sum of the first pulse width and a predetermined value included inthe detection signal.
 4. The analysis device according to claim 1,wherein the counting unit counts the particles and determines that aparticle count is one when the pulse detector detects a pulse wavehaving a pulse width greater than or equal to the first reference valueand less than a second reference value.
 5. The analysis device accordingto claim 4, wherein the second reference value is a sum of a secondpulse width and a predetermined value included in the detection signal.6. The analysis device according to claim 4, wherein, when the pulsedetector detects the pulse wave having the pulse width less than thefirst reference value and subsequently detects the pulse wave having thepulse width greater than or equal to the first reference value and lessthan the second reference value, the counting unit does not implementcounting processing with regard to the pulse wave having the pulse widthless than the first reference value detected first.
 7. The analysisdevice according to claims 4, wherein, when the pulse detector detects apulse wave having a pulse width greater than or equal to the secondreference value, the counting unit does not implement countingprocessing with regard to the pulse wave having the pulse width greaterthan or equal to the second reference value.
 8. An analysis methodcomprising: optically scanning a surface of a substrate to whichparticles are fixed; detecting a pulse wave and a pulse width of thepulse wave included in a detection signal obtained by scanning thesubstrate; and counting the particles based on a pulse interval betweentwo pulse waves each having a pulse width less than a first referencevalue when the two pulse waves are consecutively detected in thedetection signal.